Core 1: Genetically-Defined Microbe Core

Director: John Kappes, Ph.D.

Co-Director: Charles O. Elson, M.D.

Access Requests from Center investigators will be accepted on a first-come, first-served basis, subject to the requirement that the research be appropriate for the Center project and likely to contribute to its success.

Proteins are resuspended at 0.2 mg/ml in 1mM Tris, 0.1% SDS. The proteins are then be printed onto FAST 16pad slides (Whatman) using a MicroGrid II robot (Genomic Solutions). The printed slides are allowed to air-dry over night, then blocked with Protein Array Blocking Buffer (Whatman). Individual pads on the array are incubated with sera and secondarily with Alexa 647-anti-human IgG and Alexa 546- anti-human IgA. The resulting fluorescent slides are analyzed using a Genepix 4000B dual laser microarray reader (Molecular Devices) and Genepix Pro 6.0 software.

The software determines the net median pixel intensities for each individual feature (antigen spot) from a set of 10 measurements/feature. A median net digital fluorescence unit (DFU) for each feature represents the median values from 8 replicate antigen features on each array. A software program called “Significance Analysis of Microarrays” (SAM) (14, 23), which was developed at Stanford University will be applied to the data to identify antigens with statistically significant differences in antibody reactivity between groups. SAM computes a statistic for each feature, measuring the strength of the relationship between the feature intensity and the group. The software estimates a false discovery rate (FDR), i.e., a false positive rate, for each antigen by permuting the repeated measurements between groups. SAM results can be further analyzed using software called Cluster and Cluster results can be displayed using Tree View.

Packaging of the vector and cryopreservation of the virus vector stock (preparation of DNAs for packaging and Env constructs; transfection of mammalian cells; collection and preparation of culture supernatant [virus vector]; cryostorage).